Chemical crosslinkers have been used to study the molecular organization of cell membranes and to understand the way in which various molecules interact with one another at the inner or outer surface of the membrane (Peters, K., Richards, F. M., Ann. Rev. Biochem. 46:523-51 (1977), incorporated herein by reference). Protein structural studies utilizing chemical crosslinking began during the 1950s with the work of Zahn (Angew. Chem. 67:561-572, 1955; Makromol. Chem. 18:201-216, 1955; Makromol. Chem. 72:126-152 (1958), incorporated herein by reference) and continued in the 1960s, primarily with the work of Wold and his colleagues (J. Biol. Chem. 236:106-111 (1961), incorporated herein by reference). In addition, crosslinkers have been used to artificially crosslink and stabilize tissue (Nimni, M., Biorheology, 17:51-82 (1980)). Crosslinking techniques for the membrane system studies discussed above have made use of bifunctional reagents, which are classified as either homo- or heterobifunctional. Homobifunctional reagents have two identical reactive sites. Heterobifunctional reagents carry two dissimilar binding sites, one photosensitive and one conventional site. In general, both types of bifunctional reagents act to form chemical crosslinks by introducing bridges between amino acid chains.
The utility of the homobifunctional reagents as crosslinkers in membrane studies has been limited due to several potential inherent problems including random collisional crosslinks, long reaction time, difficulty in controlling reactions and nonselective crosslinking. Random collision dependent crosslinks can occur at a significant frequency, since molecules nonspecifically crosslink during random collisions in fluid membranes. Such indiscriminate formation of crosslinks can result in a high multiplicity of crosslinked products which are difficult to analyze. It is possible, therefore, that low yield crosslinked products would go undetected. These random collisional crosslinks were avoided in some membrane systems with the use of rapidly crosslinking photosensitive agents. (Ji, T. H., Biochimica et Biophysical Acta, 559:39-69 (1979), incorporated herein by reference).
In contrast, crosslinking with photosensitive heterobifunctional reagents, can be easily, rapidly and sequentially controlled. Crosslinking with heterobifunctional reagents is accomplished by binding the conventional site on the reagent to one amino group via an amide bound, leaving the second photoactivatable site unbound. Upon photoactivation by the use of ultraviolet or visible irradiation, the photoactivatable site is converted to a species of very high chemical reactivity, which then forms a covalent linkage with another amino group. The absorption of ultraviolet or visible radiation by the bifunctional reagent can give rise to two general classes of species produced by cleavage of chemical bonds. Fragmentation can be either at a single bond, resulting in the formation of two free radicals, or at a double bond to carbon or nitrogen. Two types of photosensitive groups are known that result from cleavage at a double bound to carbon or nitrogen: an azide derivative and a diazo derivative. Nitrenes are generated from azides, and carbenes are generated upon photolysis of diazo derivatives. Both nitrenes and carbenes are compounds of very high chemical reactivity.
A common method used for photoactivation of heterobifunctional compounds is irradiation with a short wave ultraviolet lamp, for example, mineral light USV-11. The half time of photolysis with this lamp varies depending on the reagents and is in the order of 10 to 50 seconds. An alternative method, which has several advantages, is flash photolysis for an extremely short period, normally on the order of milliseconds.
Collagen is the single most abundant animal protein. It is the main structural component of mammalian tissues and accounts for about 30% of all mammalian proteins (Nimni, M., Biorheology, 17:51-82 (1980), incorporated herein by reference). The molecular structure of collagen consists of three intertwining helical polypeptide chains about 1,050 residues long, wound around each other to form a triple helix.
There is a great amount of uniformity in the amino acid composition of collagen. Glycine forms about 33 percent and proline and hydroxyproline form about 25 percent of the total amount of residues in the polypeptide chains. Proline and hydroxyproline contribute to the rigidity of the molecule in that the beta C is linked to the peptide nitrogen by the side chain, forming a five membered ring thus allowing relatively little freedom of rotation. It is this locking effect by proline and hydroxyproline residues, and the hydrogen bond formation by the hydroxyl group of hydroxyproline, which gives collagen its great stability. The other amino acid residues in the structure include 10 percent alanine and 20 percent polar side chains of arginine, lysine, asparagine and glycine. These do not play a particularly important role in the triple helix but nevertheless are important in the intermolecular linkages which lead to fiber formation.
Crosslinking of the collagen molecules occurs extracellularly and leads to formation of the collagen fiber. This characteristic fiber organization is responsible for the functional integrity of tissues such as bone, cartilage, skin and tendon, and for the structural integrity of blood vessels and most organs.
Both intra- and intermolecular crosslinks in collagens are derived from lysine and hydroxylysine residues. Intramolecular crosslinks are formed when specific lysine and hydroxylysine residues in collagen are oxidatively deaminated to peptide bound aldehydes. Copper, a cofactor with the enzyme lysyl oxidase, causes this modification to take place. The actual formation of the crosslinks takes place via aldol condensation, a spontaneous non-enzymatic reaction where the lysines which are located near the end-terminal region are converted to aldehydes. Intermolecular crosslinks are formed between peptide bound aldehydes and unmodified amino groups of other lysine and hydroxylysine residues. These are the Schiff base type crosslinks, otherwise known as aldamine crosslinks (aldehyde and amino group). This type of crosslink is also considered to be the most physiologically important.
Crosslinking of collagen is a prerequisite for the collagen fibers to withstand the physical stresses to which they are exposed. In past investigations, chemical agents, in particular glutaraldehyde, were found to have application for biosynthesis of intramolecular and intermolecular crosslinks. Artificial crosslinking of collagen with glutaraldehyde has been used commercially to stabilize pig heart valves which are then used in artificial valve replacements (Nimni, M., Biorheology, 17:51-82 (1980), incorporated herein by reference). The collagen is crosslinked in this technique with 25 percent glutaraldehyde (commercial) at a neutral pH. The exact glutaraldehyde chemistry of the crosslinking is not clear but Schiff base linkages of glutaraldehyde with two lysine residues are formed.
Many studies have been conducted to develop a substance, either natural or synthetic, which can be employed as a non-traumatic means to help repair tissues after surgery. Major interest in the surgical use of polymeric adhesive materials began in the early sixties (Silverstone. et al., Arch. Surg. 81:98 (1962), incorporated herein by reference). Initial work was confined to water-soluble systems such as casein and polyvinyl alcohol, but later was expanded to include all available synthetic adhesives and other plastics. Effort at this point was limited to materials with no known local or general toxicity. The 1962 effort of Silverstone and his coworkers was directed more towards wider application of bonding techniques in arterial surgery. In addition to the reinforcement of aneurysms unsuitable for resection, the uses contemplated included reinforcement of junctions after arterial suture or graph, and non-suture anastomosis of small arteries. Although other materials have been investigated, the most widely used of the tissue adhesives are the cyanoacrylates. These are a homologous series of organic molecules which polymerize and adhere to moist living tissues. Methyl-alphacyanoacrylate (MCA) in particular, has been used since 1960 by many investigators as a tissue adhesive for non-suture of bones. MCA is a fluid, monomeric material which under mild pressure, polymerizes in a matter of seconds to produce a thin, strong, adherent film. Although MCA has been shown to be histotoxic, work with higher homologues of the n-alkyl-alphacyanoacrylates has indicated that if one proceeds up the homologous series, this histotoxicity decreases. The toxic effects of synthetic polymers on tissues are related in part to their breakdown products and to the rate at which they are released. All of the polycyanoacrylates degrade in an aqueous medium by the same mechanism--the cleavage of the carbon-to-carbon backbone of the polymer, and the ultimate releasing of formaldehyde and other breakdown products. This mechanism of degradation is essentially the same for all the alkyl cyanoacrylates, though the rate is quite different and depends on the nature of the radical.
It has been reported that the less toxic higher homologues of the cyanoacrylates instantaneously polymerize on tissue substrates and thereby are more effective in inducing homeostasis. Instantaneous polymerization, however, is a disadvantage in surgical applications where it is required to bond two surface's together, or in adhering cut surfaces of an organ. In these instances, one requires sufficient working time to approximate the surfaces of the tissues before adhesion is permitted to take place.
In order to accommodate these surgical requirements, application techniques of tissue adhesives have been investigated (Matsumoto, T., Tissue Adhesives Insurgent, Med Exam. Pub. Co., New York (1972), incorporated herein by reference). Tissue adhesives were applied using a spray gun or by a drop method. Polymerization of the adhesive occurred more rapidly when it was applied by spraying. The difference in rates of polymerization was explained by the fact that on spraying, the monomers formed a spreading film, making more surface available to the initiator and thereby a more rapid polymerization rate.
In many surgical techniques the use of the spray method discussed above has a distinct advantage because it is not possible to apply the monomer uniformly and in a thin film with the drop method. Spraying, however, has one disadvantage, in that the monomer polymerizes more rapidly and makes it necessary for the surgeon to work faster. The advantages of and need for an adhesive wherein the surgeon can control the polymerization rate is therefore clear.
In addition, although the reports indicate that cyanoacrylate tissue adhesives offer advantages when used for repair or homeostasis of injured organs, it is known that the presence of the polymer fragment between the incised skin delays wound healing. This is because the polymer fragments prevent the proliferation of fibroblast and microcirculatory vessels bridging the wounded surfaces. Studies conducted comparing the tensile strength of wounds closed by sutures versus cyanoacrylate adhesives, have shown that the glue remains in the tissue for long periods of time, and maximal wound strength is obtained later than for suture closure.
Application of cyanoacrylate adhesives in ophthalmological procedures was introduced in 1963 (Bloomfield, S. et al., Amer. J. Ophthal., 55:742-748 (1963), incorporated herein by reference). Since the maintenance of a delicate metabolic and pressure balance within the eye is vital to its optical and electrophysiological function and depends on the integrity of the outer coat, considerable attention in ophthalmology has always been directed towards methods of repair of any process which disrupts the cornea or sclera. Early experience with cyanoacrylate adhesives in the eye was not particularly encouraging. Methyl-2-cyanoacrylates were found to have suitable bond strength, but they proved too toxic.
Over the past century, a number of substances other than the cyanoacrylates have been proposed for sticking one tissue to another, but as with the cyanoacrylates, none appear to have been entirely successful.
Crosslinked gelatins are a leading contender with the cyanoacrylates for the attention and interest of investigators working on tissue bioadhesives. Gelatin is a naturally occurring animal protein with innate adhesive properties. Molecular weights of gelatins range between 30,000 and 120,000 and chemically it is somewhat similar to connective tissue. In 1965, Braunwald and Tatooles (Surgery, 19:460 (1946), incorporated herein by reference) reported the successful use of crosslinked gelatin to control hemorrhage from wounds of the liver and the kidney in dogs. Still later, Bonchek and Braunwald (Ann. Surg., 165:420 (1967), incorporated herein by reference) also describe the use of crosslinked gelatin to repair incisions in dogs. The main problem with gelatin as a bioadhesive however, is that it is highly susceptible to enzymatic degradation.
Other substances with some adhesive properties have been used to help ocular wounds heal quickly and firmly. Parry and Laszlo reported the use of thrombin for a quick and efficient sealing of conjunctival wounds in corneal scleral incisions in cataract surgery (Brit. J. Opthal., 30:176-178 (1946), incorporated herein by reference). Town used fibrin in cataract, glaucoma and traumatic plastic surgery and in keratoplasty (Trans. Amer. Acad. Ophthal. Otolaryng., 54:131-133, (1949), incorporated herein by reference). But Young and Favata pointed out that thrombin imparts less tensile strength than ordinary suture materials (War. Med., 6:80-85 (1944), incorporated herein by reference). Another adhesive that has been investigated is fibrinseal (FS) which is a natural adhesive material composed of fibrinogen, factor VIII, platelet growth factor, anti-plasmin thrombin, and calcium chloride. FS has been utilized in vascular surgery to limit blood loss and minimize the amount of vessel trauma and foreign-body reaction by decreasing the number of sutures necessary to achieve a technically satisfactory arterial anastomosis. However, FS causes an increase in the amount of lymphocytic infiltrate in specimens early in the post operative period. As the authors admit, detailed studies to define its role and drawbacks are in order (Ikeossi-O'Connor, M. G., Journal of Surgical Oncology, 23:151-152 (1983), incorporated herein by reference).
A human fibrin glue has been used in oral surgery (Bull. Group. int. Rech. sc. Stomat. et Odont., 27:171-180 (1984), incorporated herein by reference). The substance is made up of two components. One, is highly concentrated fibrinogen and factor VIII together with other plasma proteins, such as albumin and globulin. The second component is a solution of thrombin and calcium chloride, a catalytic agent. The Factor VIII induces the collagen present in the connective tissue to polymerize with the fibrin, forming a bridge between collagen and fibrin. Some known disadvantages of this fibrin glue are that once prepared, it must be used within a short time (so the surgeon must possess accuracy and speed in the operating technique), and the possible transmission of the hepatitis and AIDS viruses.
The foregoing discussion describes the efforts to use a variety of substances of both natural and artificial origin as tissue adhesives. None of these efforts have been completely successful. There still remains both a need for, and a desire for, a tissue adhesive which is simple and practical in application, which is not toxic, which does not retard wound healing, which is readily and harmlessly absorbed and eliminated to normal metabolic pathways once it is served its purpose, and which is without carcinogenic or any other harmful long range potential problems.
The following is a list of desirable criteria for bioadhesives, one or more of which has not been met by the prior materials.
1. Ease of application. PA1 2. Control of polymerization. PA1 3. Flexibility of the resulting bond. PA1 4. Bond strength. PA1 5. Transparency. PA1 6. Low toxicity. PA1 7. Biodegradability.